Project Summary: Alcohol consumption is widespread in the United States with ~7% of alcohol-consuming individuals engaging in heavy alcohol use. It is well established that chronic heavy drinking (CHD) is associated with increased susceptibility to infections as well as impaired wound healing and tissue repair resulting in poor post-operative outcomes. Evidence suggests that many of these defects are mediated by excessive inflammatory responses originating from myeloid cells, notably circulating monocytes and tissue-resident macrophages. However, many of the current studies rely on in vitro exposure of monocytes from healthy donors or cell lines to high doses of ethanol. Due to a lack of studies utilizing reliable in vivo models, our understanding of the mechanisms underlying aberrant inflammatory responses in the context of CHD remains incomplete. In order to address these knowledge gaps, we propose to leverage a rhesus macaque model of voluntary ethanol self-administration that accurately mirrors human physiology and recapitulates complex human drinking behavior. Using this model, our lab has recently demonstrated that CHD results in transcriptional and epigenetic rewiring of circulating monocytes and splenic macrophages, resulting in aberrant responses to LPS stimulation. However, the functional implications of and the epigenetic mechanisms controlling this reprogramming remain unknown. Importantly, because monocytes are short-lived circulating cells under constant repopulation from the bone marrow, these observations suggest perturbations of the hematopoietic niche. Preliminary single-cell analyses of hematopoietic progenitors point to a shift in differentiation potential towards more mature myeloid progenitors with alcohol. However, a link between this phenotype in progenitor cells and their differentiated states in blood remains unclear. In this application, we propose to test the hypothesis that chronic alcohol consumption reprograms the epigenetic landscape of monocyte progenitors in the bone marrow giving rise to circulating monocytes poised towards a hyper-inflammatory response. We will first examine the impact of CHD on functional reprogramming of circulating monocytes, implementing assays to test their ability to migrate, phagocytose, and generate proper metabolic responses. We will then examine the effect of stimulation on the monocyte epigenetic landscape through assessment of chromatin accessibility and differential binding of histone modifications with alcohol. Finally, we will determine the effect of CHD on the differentiation potential, transcriptome activation, and epigenetic rewiring of bone marrow myeloid progenitors. We will perform functional assays on monocytes derived in vitro from granulocyte/monocyte progenitors and integrate these data with those obtained from peripheral monocytes. Further, scRNA-Seq analysis and epigenetic assessment of the myeloid progenitors will allow us to determine the specific effects of alcohol on the bone marrow compartment and how this leads to hyper-inflammatory, epigenetically reprogrammed peripheral monocytes. Completion of this proposal will expand our knowledge of the immunological effects of alcohol consumption on hematopoiesis.